Compositions and methods for introducing effectors to pathogens and cells

ABSTRACT

Compositions and methods for the inhibition and prevention of pathogenic infection and neoplastic disease are provided. The compositions include hybrid molecules having a binding moiety and an effector moiety joined by a linker region. When administered to a host, the binding moiety, such as a carbohydrate, attaches to a receptor, such as a conserved lectin receptor on the pathogen or neoplastic cell, and the effector moiety provides an invariant antigenic determinant for eliciting or modulating an immune response. The effector moiety may also be a drug or other compound which inhibits growth of a bound pathogen or cell. Compositions comprising the hybrid molecule in a suitable pharmaceutical carrier are also provided.

This invention was made with Government support under Grant (orContract) No. NIH 1946002123A1, awarded by the Department of Health andHuman Services. The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compositions and methods forspecifically targeting pathogens and cells. More particularly, thepresent invention relates to the use of hybrid molecules including areceptor-binding moiety and an effector moiety for altering theantigenic character of or delivering drugs to pathogens anc cells.

The primary defense mechanism of man and other vertebrates againstpathogenic infection is the immune system. The immune response includestwo separate pathways to deal with invasion by a foreign substance. Thefirst pathway, referred to as the "humoral response," relies on antibodymolecules to bind directly to pathogen to trigger a series of events(the complement cascade or the binding of macrophages and otherleukocytes) to eliminate the pathogen from the body. The second immunepathway, referred to as the "cellular response," relies on T-cellrecognition of an antigenic region on the pathogen, again leadingultimately to elimination of the pathogen from the system. Both thehumoral and cellular responses thus rely on antigenic recognition of thepathogen in order to kill the pathogen and protect the host.

While the immune response is an exquisite and effective protectivemechanism against a wide variety of pathogens, there are certainpathogens which evade both pathways of the immune response by changingtheir cell surface antigens sufficiently rapidly so that they are notrecognized by the antibodies which have been elicited during earlierstages of infection. Pathogens with such an ability to evade the immuneresponse include viruses, such as the influenza virus, papillomaviruses, picornaviruses, polyoma virus, and rhinoviruses; bacteria, suchas Escherichia coli and Vibrio cholerae; and protozoa, such as Entamoebahistolytica, Trypanosoma cruzii, Plasmodium knowlesi, P. vivax and thelike.

Most or all pathogens initiate infection by binding to a surface ligandon the cell being infected. For example, the pathogen may possess alectin receptor which is able to specifically bind to a carbohydrateligand on the cell to be infected. To prevent infection, it has beenproposed to inhibit initial attachment of the pathogen using drugs whichblock binding of the pathogen to the cells which are subject toinfection. While such drugs can be effective, high dosages may berequired to block all available binding receptors on the pathogen.Moreover, the drugs are passive and do not enhance the killing andelimination of the pathogen from the host. Another class of blockingagents include soluble polypeptide receptors, such as soluble CD4 usedto inhibit binding of HIV-1 to T-cells. The use of soluble polypeptidereceptors has not generally been successful, perhaps due to degradationof the polypeptides after they are administered to a patient.

Sialic acids, derivatives of N-acetyl neuraminic acid (NeuAc), arecarbohydrate groups found terminating cell-surface glycoproteins andglycolipids. Glycosides of NeuAc are often utilized by pathogens as anattachment point to cells prior to infection. The use of sialic acidanalogs as drugs directed towards the influenza virus has been proposed.The use of O-linked glycosides as potential viral inhibitors, however,is severely limited because of the presence of the neuraminidase enzymeon the virus. This enzyme cleaves the glycosidic bond of NeuAc givingrise to the free sugar which does not inhibit viral attachment.Therefore, a stable non-hydrolyzable analog of sialic acid promises tobe useful as an antiviral drug.

For these reasons, it would be desirable to provide improvedcompositions and methods for enhancing a host's immune response againstpathogenic infection, particularly against pathogenic infection byorganisms capable of altering their antigenic appearance over time. Itwould be particularly desirable to provide compositions which are ableto target a pathogen and provide at least one invariant antigenicdeterminant so that a host's immune response can target the pathogenbased on the invariant determinant. The compositions will desirably besmall, preferably being less than 3 kilodaltons (kD), more preferablybeing less than 2 kD, and most preferably being less than 1 kD in orderto increase their survival time after administration to the host. Thecompositions should further be substantially free from non-specificbinding so that they target the immune response solely against thedesired pathogenic organism. The compositions will preferably notthemselves be destroyed by the immune response so that individualmolecules may successively bind more than one pathogen to reduce thedosage required. It will further be desirable to administer compositionswhich elicit a secondary or memory response against an antigen againstwhich the host has been previously sensitized.

2. Description of the Background Art

Soluble hybrid molecules, designated immunoadhesins, comprising thegp120-binding domain of CD4 glycoprotein attached to portions of the Fcregion of IgG are described in Capon et al. (1989) Nature 337:525-531.See also European Patent Application 0 314 317. Use of theimmunoadhesins for treatment of acquired immunodeficiency syndrome(AIDS) is proposed. Hybrid receptors comprising the ligand-bindingdomain of a receptor, such as a growth factor receptor, attached to aheterologous reporter polypeptide, such as an enzyme are described inU.S. Pat. No. 4,859,609, to Dull and Ullrich. Schultz and Shokat (1991)J. Am. Chem. Soc. 13:1861 describe the use of CD4-nitrophenol conjugatesto target anti-DNP antibodies against HIV-1. Sharon and Lis (1989)Science 246:227-234 describe the nature of some pathogenic receptors(lectins) which bind to cell surface carbohydrates to initiateinfection. Win compounds are described in Badger et al. (1988) Proc.Natl. Acad. Sci. 85:3304-3308. Win compounds bind to rhinoviruses andinhibit uncoating of virus (which is necessary for infection). Thesynthesis of sialic acid analogs intended for use as drugs is reportedin Sauter et al. (1989) Biochemistry 28:8388 and Whitesides (1991) J.Am. Chem. Soc. 113:686-687.

SUMMARY OF THE INVENTION

The present invention comprises a hybrid molecule which is able to bindto a receptor, usually a conserved receptor, on a pathogen or neoplasticcell in order to introduce at least one heterologous determinant siteonto the pathogen or cell surface. The hybrid molecules are smallmolecules, typically having molecular weights below about 3 kD, whichcomprise a binding moiety attached to an effector moiety capable ofeliciting or modulating an immune response when administered to a host.The binding moiety preferably binds to the pathogenic or cellularreceptor with an affinity of at least about 1 mM⁻¹ (10⁻³ M⁻¹). Thebinding moiety will typically mimic a carbohydrate binding regionpresent on a host cell which binds the pathogen receptor as part of theinitiation of infection or a neoplastic cell as part of the metastaticprocess. Usually, the binding moiety will include at least one sugarcharacteristic of the host cell binding ligand (usually a terminalsugar) joined to the remainder of the hybrid molecule through acarbon-linkage. Such carbon-linked sugar(s) are desirable since they areless subject to chemical and enzymatic degradation, than are naturaloxygen-linked sugars. Frequently, more than one sugar from the cellsurface ligand will be incorporated in the hybrid molecule.Alternatively, other small drugs and moieties capable of binding to thereceptor on the pathogen or neoplastic cells, other than carbohydrates,may be employed.

The effector moiety will usually be capable of eliciting, modulating, orotherwise participating in the humoral immune response when administeredto a host as part of the hybrid molecule. In particular, the effectormoiety may elicit a primary immunogenic response when the host has notbeen previously sensitized to the antigenic determinant defined by theeffector. Alternatively, and preferably, the effector moiety may elicita secondary or memory response when the host has previously beensensitized to the corresponding antigenic determinant. In addition or asa further alternative, the effector moiety may be a drug capable ofkilling a bound cell, inhibiting cellular attachment, or otherwiseinterfering with the pathogenic or metastatic process.

The linker will be capable of covalently binding to both the bindingmoiety and the effector moiety and will be able to maintain the properspacing therebetween, typically from about 10 Å to 40 Å. The linker willusually be flexible to permit relative motion between the binding moietyand the effector moiety so that the effector moiety will be sufficientlyexposed on the surface of the pathogen to participate in the desiredinteraction with the cells of the host's immune system. The linkershould be substantially free from any features which could result innon-specific binding of the hybrid molecules.

The present invention further comprises compositions containing thehybrid molecules as just described present in apharmaceutically-acceptable carrier.

The present invention also comprises novel carbon-linked sialic acidderivatives which may be particularly useful in preparing the hybridmolecules described above.

The present invention provides methods for introducing a heterologousantigenic determinant to a pathogen or neoplastic cell by exposing thepathogen or cell to the hybrid molecules described above. The bindingmoiety of the hybrid molecules will specifically bind to the receptor,usually a conserved receptor, on the pathogen or cell, and the effectormoiety provides an invariant determinant which can act as a target toelicit an immune response against the pathogen. The effector may alsoprovide for direct killing of the pathogen or cell. In a particularaspect, the method can be used to treat pathogenic infection byadministering the hybrid molecule to a host to elicit or modulate thehost's own humoral or cellular immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4 illustrate the synthetic schemes for the preparation ofcarbon-linked mannose glycosides as described in the Experimentalsection hereinafter.

FIG. 5 is an electron micrograph showing the localization of goldparticles on the surface of an E. coli cell. The particles were boundthrough a hybrid molecule consisting of a mannose binding moietyattached to biotin through a carbon linkage. Avidin, anti-avidinantibodies, and protein A labelled with the gold particles were exposedto cell and completed a bridge binding the gold particles. The locationof a gold particle is shown by the arrow.

FIG. 6 is an electron micrograph showing a strain of E. coli, similar tothat illustrated in FIG. 5, in the absence of the hybrid molecule. Thecell was exposed to avidin, anti-avidin antibodies, and protein Alabelled with gold particles. No localization of the gold particles wasachieved without the hybrid molecules.

FIGS. 7-9 illustrate the synthetic scheme for the preparation of acarbon-linked sialic acid residue as described in the Experimentalsection hereinafter.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

According to the present invention, hybrid molecules include a bindingmoiety and an effector moiety covalently joined together by a linkerregion. The binding moiety is capable of specifically binding to aconserved surface receptor, typically a lectin, on a pathogen ofinterest while the effector moiety is capable of eliciting or modulatingan immune response when administered to a host. The linker is selectedto maintain the effector molecule in a proper position relative to thepathogen to permit interaction with the host's immune system when thehybrid molecule is bound to the pathogen.

The hybrid molecules of the present invention are used to"immunologically target" a wide variety of human and animal pathogensincluding viruses, bacteria, and protozoa. An exemplary list ofpathogens which may be targeted is set forth in Table 1.

                  TABLE 1                                                         ______________________________________                                                            Binding Sugar.sup.1                                       Pathogen            or other Moiety                                           ______________________________________                                        VIRUSES:                                                                      Influenza Virus     NeuAcα2→6Gal                                                     NeuAcα2→3Gal                                 Picornaviruses      Win compounds.sup.2                                       Polyoma Virus       NeuAcα2→6Gal                                 BACTERIA:                                                                     Escherichia coli                                                              Type 1              Man(α-glycosides)                                   Type P              Galα1→4Gal                                   Type S              NeuAcα2→3Gal                                 Vibrio cholerae     Gm.sub.3 (ganglioside)                                    Actinomyces         Gal-X.sup.3                                               PROTOZOA:                                                                     Entamoeba histolytica                                                                             Galβ1→4GlcNAc                                 Plasmodium knowlesi Duffy antigen                                             Plasmodium vivax    Duffy antigen                                             Trypanosoma cruzi   NeuAc.sup.3                                               ______________________________________                                         .sup.1 NeuAc: Nacetylneuramic acid (sialic acid)                              Gal: Galactose                                                                GlcNAc: NAcetylglucosamine                                                    Man: Mannose                                                                  .sup.2 Win compounds are described in Badger et al. (1988), supra., the       disclosure of which is incorporated herein by reference. These compounds      specifically bind to a pocket within the viral protein VP1 β-barrel      structure and are useful as binding moieties in the compositions of the       present invention.                                                            .sup.3 Exact structure undetermined.                                     

The target pathogen may be any pathogen which has a receptor, preferablya conserved receptor, which binds to a cell surface ligand as part ofthe initiation of infection within that cell. The pathogen receptors areusually lectins but may also be other molecules, such as glycosidicenzymes, and the cell surface ligands are usually carbohydrates in theform of glycoproteins, glycolipids, oligosaccharides, andpolysaccharides. Upon exposure to the pathogen, the binding moiety ofthe hybrid molecule will specifically attach to the pathogen receptor.The length and nature of the linker region permits the effector moietyto appear as an antigenic determinant of the pathogen itself. Thus, thepathogen will be processed by the host's immune system as if itnaturally possessed the antigenic determinant defined by the effectormoiety. The present invention is particularly useful for targetingpathogens having antigenic characteristics which vary over time, wherethe introduced antigenic determinant is invariant and can elicit ormodulate a sustained immune response.

The hybrid molecules of the present invention will also be useful forbinding and treating neoplastic cells based on binding of cell ligandsto lectin receptors present on the neoplastic cells. Exemplary lectinreceptors on neoplastic cells are responsible for binding tocarbohydrate ligands on vascular endothelial cells, which is a necessarymechanism in the metastatic process. Thus, by employing binding moietieswhich mimic these cell-surface carbohydate ligands, present on theendothelial cells, the hybrid molecules can specifically attach to theneoplastic cells. A number of exemplary carbohydrate ligands andcorresponding neoplastic cell receptors are identified in Raz et al.(1987) Can. Met. Rev. 6:433-452, the disclosure of which is incorporatedherein by reference. Specific extracellular matrix adhesion moleculesare described in McCarthy et al. (1985) Can. Met. Rev. 4:125-152; thecell-cell CAM adhesion system is described in Brackenbury (1985) Can.Met. Rev. 4:41-58; and the lymphocyte homing receptor system isdescribed in Stoolman (1989) Cell 56:907-910, the disclosures of whichare incorporated herein by reference.

The binding moiety will be selected to specifically bind to a receptor,usually a conserved receptor, on the pathogen or neoplastic cell ofinterest. The moiety will typically have a molecular weight below about2 kD, usually being between about 2 kD and 400 daltons, preferably beingbetween about 1.5 kD and 500 daltons, and will usually be other than apolypeptide or protein.

Most commonly, the binding moiety will be a carbohydrate comprising oneor more sugars which are selected to mimic sugars, usually the terminalsugars, on the carbohydrate binding ligands on the cells infected by thepathogen (or attached by the neoplastic cell), usually being identicalto the terminal residues which are bound during the infection ormetastatic initiation process. Exemplary sugars include mannose, sialicacid, galactose, fucose, α-glucosamine, and derivatives thereof, usuallylinked through carbon rather than oxygen. Specific terminal sugars for avariety of pathogens are set forth in Table 1 above.

Other pathogens having such conserved carbohydrate ligands are describedin the medical and scientific literature. See, for example, Cornfield etal., in Sialic Acids, Schauer, Ed., Springer-Verlag, New York, 1982.McGuire, in Biological Roles of Sialic Acid, Rosenberg and Schengrind,eds., plenum, New York, 1976; Sharon and Lis, Lectins, Chapman and Hall,London, 1989; and Mirelman, ed., Microbial Lectins and Agglutinins:Properties and Biological Activity, Wiley Series in Ecological andApplied Microbiology, Wiley-Interscience Publication, John Wiley andSons, New York, 1986, the disclosures of which are incorporated hereinby reference.

Carbohydrate binding moieties may be monovalent or multivalent and willusually comprise from one to eight sugars, more usually comprising fromone to four sugars, and preferably comprising from one to two sugars,with the individual sugars being joined by the glycosidic linkages whichare present in the natural carbohydrate ligand, i.e., maintaining thesame stereochemistry, preferably employing carbon linkages instead ofthe oxygen linkages. Methods for synthesizing such saccharides andoligosaccharides are well described in the chemical literature. See, forexample, Paulsen and Tietz (1985) Agnew. Chemie. Intl. Ed. Engl. 24:128;Toone et al. (1987) Tet. Lett. 44:5365-5422; Palcic et al. (1989) Carb.Res. 190:1-11; Sabesin et al. (1986) J. Am. Chem. Soc. 108:2068-2080;and Schmidt (1986) Agnew. Chemie. Intl. Ed. Engl. 25:212, thedisclosures of which are incorporated herein by reference. A particularmethod for synthesizing a carbon-linked sialic acid (NeuAc) is describedin detail in the Experimental section hereinafter.

In addition to carbohydrate binding moieties as just described, thepresent invention may employ other small synthetic moieties that have aadequate binding affinity and specificity to the conserved receptors onthe pathogen or neoplastic cells of interest. In particular, the bindingmoiety (carbohydrate and non-carbohydrate) should have a bindingaffinity to the pathogen or cellular receptor of at least about 1 mM⁻¹(10⁻³ M⁻¹), preferably being at least about 0.01 mM⁻¹ (10⁻⁵ M⁻¹), andmore preferably being at least about 1 μM⁻¹ (10⁻⁶ M⁻¹).

The non-carbohydrate binding moieties will usually be other than aprotein or polypeptide. Exemplary non-carbohydrate binding moietiesinclude the Win compounds described in Badger et al. (1988) supra.

The identification of other compounds that can be employed as thebinding moiety, i.e., those which are capable of binding to the pathogenreceptor with the requisite affinity, can be achieved through the use oftechniques known to those working in the area of drug design. Suchmethods include, but are not limited to, self-consistent field (SCF)analysis, configuration interaction (CI) analysis, and normal modedynamics computer programs, all of which are readily available. See,Rein et al., Computer-Assisted Modeling of Receptor-Ligand Interactions,Alan Liss, New York, (1989).

Preparation of the identified compounds and moieties will depend ontheir structure and other characteristics and may normally be achievedby standard chemical synthesis techniques. See, for example, Methods inCarbohydrate Chemistry, Volumes I-VII: Analysis and Preparation ofSugars, Whistler et al., eds, Academic Press, Inc., Orlando, 1962, thedisclosure of which are incorporated herein by reference.

The effector moiety will be selected to provide a desired response orrecognition by the host's immune system (usually humoral) when themoiety becomes attached to the pathogen or neoplastic cell as a resultof the hybrid molecule binding to a receptor associated with thepathogen or cell. The effector moiety itself will frequently be a smallcompound, typically having a molecular weight below about 1 kD,preferably below about 500 D, usually in the range from about 100 to 400D, and more usually in the range from about 250 to 350 D. Thus, theeffector moiety will often be haptenic rather than antigenic, i.e., itwill be unable to induce a primary immune response by itself but will beimmunogenic when combined with the remainder of the hybrid molecule.Even small (haptenic) molecules will, however, induce a secondary ormemory response in hosts who have been previously sensitized to theparticular antigenic determinant.

Under certain circumstances, it may be desirable to employ largercompounds as the effector moiety, typically having molecular weightsabove about 1 kD, sometimes having molecular weights above about 1.5 kD,and occasionally having molecular weights above about 2 kD. Suchantigenic compounds will usually be polypeptides or proteins, willusually be immunogenic by themselves (i.e., without binding to theremainder of the hybrid molecule) and may define more than one antigenicdeterminant site.

Antigenic determinants, sometimes referred to as epitopic sites orepitopes, are the portion of an antigenic or haptenic molecule thatinteracts with the host's immune system by molecular complementarity. Inthe humoral response, it reacts with cell surface Ig on B-cells toinduce antibody production.

The antigenic determinant site(s) defined by the effector moiety willpreferably be cross-reactive with an antigen or hapten to which the hosthas been previously sensitized. In this way, administration of thehybrid molecule to the host will evoke a secondary or memory responsewhich will have a substantially immediate effect on the target pathogenor neoplastic cells. Alternatively, the effector moiety may be selectedto introduce an antigenic determinant to which the host has not beenpreviously exposed. In the latter case, the determinant will evoke aprimary humoral response when administered to the host.

Exemplary effector moieties include those capable of producing a strongimmunogenic response when administered to the host as part of the hybridmolecule, e.g., aminophenols, nitrophenols, fluorescent probes(fluroscein), phenolic glycosides (p-aminophenol-β-glycoside), and thelike. Preferred effector moieties for which the host has a naturalimmunity include blood group carbohydrates, e.g.,α-D-GalNAc(1→3)β-D-Gal, α-D-Galp-(1→3)-β-D-Gal,α-D-GalpNAc-(1→3)-β-D-Gal, and α-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Gal;dinitrophenol (DNP); Galα1-3Gal; and the like.

A particularly preferred effector moiety is the Galα1→3Gal disaccaridefor which most individuals have a natural immunity. The disaccharide andits properties are described in Galili et al. (1987) Proc. Natl. Acad.Sci. USA 84:1369-1373; Galili et al. (1987) J. Biol. Chem.262:4683-4688; and Galili et al. (1986) J. Clin. Invest. 77:27-33, thedisclosures of which are incorporated herein by reference. TheGalα1→3Gal structure is preferred because it has been found that up to1% of all circulating IgG in individuals is reactive with thedisaccharide. The disaccharide can be prepared by chemical synthesis orby transfer of galactose using an appropriate glycosyltransferase to anacceptor molecule bearing a terminal galactosyl moiety. See, Larsen etal. (1989) Proc. Natl. Acad. Sci. USA 86:8227-8231, which describes aparticular method for synthesizing the Galα1→3Gal disaccharide, thedisclosure of which is incorporated herein by reference.

The hybrid molecule compositions of the present invention may alsoemploy commonly used and commercially available compounds as theeffector moiety. The use of biotin and fluoroscein effector moieties isdescribed in detail in the Experimental section hereinafter.

While the effector moiety will most commonly provide for an invariantantigenic determinant, as described above, it is also possible that theeffector moiety may be a drug or cytotoxic compound intended to kill orinhibit growth of the pathogen or neoplastic cell. Exemplary drugs andcytotoxic agents include antibacterial drugs; anti-neoplastic drugs;photoactivated compounds; radionuclides; toxin A chains, such as ricin Achain and abrin A chain; and the like.

While the hybrid molecule compounds of the present invention will findtheir greatest use in antigenic targeting of pathogens and neoplasticcells, they may also be used for the detection of pathogens orneoplastic cells in in vitro assays. The use of both biotin andfluoroscein effector moieties is particularly convenient for in vitroassays as both biotin and fluoroscein are well known reporter moleculesfor which a wide variety of detection systems exist.

The linking region or group is selected to provide the necessarycovalent bridge between the binding moiety and the effector moiety.Frequently, the linking region will be derived from a bifunctionalcompound having a reactive group at one end which is capable of bindingto the binding moiety and a second reactive group which is capable ofbinding to the effector moiety. Alternatively, the linking region may besynthesized together with either the binding moiety or the effectormoiety and will then include only a single reactive functionality forcovalent binding to the other moiety.

The nature of the linking region is not critical, but it should providea sufficient spacing and flexibility between the binding moiety and theeffector moiety so that the effector moiety is sufficiently exposed onthe surface of the pathogen to interact with the host's immune system ina desired manner. The length of the linking region will usually bebetween about 10 Å and 40 Å, preferably being between about 15 Å and 30Å. The linking region should be resistant to degradation whenadministered to a host as part of a hybrid molecule and should furthernot contribute to non-specific adhesion of the hybrid molecule, i.e.,adhesion or binding to other than the target receptor. It will beappreciated that the hybrid molecules of the present invention shouldbind with a high affinity and specificity to only the pathogen ofinterest.

The carbohydrate binding moieties will preferably be attached to theother portions of the hybrid molecule through carbon bonds. Whilenatural glycosidic linkages occur through an oxygen (O-glycosidicbonds), the hybrid molecules of the present invention preferably employcarbon-glycosidic bonds to promote stability and inhibit chemical andenzymatic hydrolysis. Specific syntheses techniques for preparing suchC-linked glycosides are set forth in the Experimental sectionhereinafter.

Exemplary bifunctional compounds which can be used for attachingcarbohydrate moieties to effector moieties include bifunctionalpolyethylene glycols, polyamides, polyethers, polyesters, and the like.General approaches for linking carbohydrate moieties to other smallmolecules, polypeptides, and the like, are well described in thechemical literature. See, for example, Lee et al. (1989) Biochemistry28:1856 (carbohydrate conjugation); Bhatia et al. (1989) Anal. Biochem.178:408 (protein conjugation); Janda et al. (1990) J. Am. Chem. Soc.112:8886 (protein conjugation), the disclosures of which areincorporated herein by reference.

The hybrid molecules of the present invention can be incorporated ascomponents of pharmaceutical compositions useful to attenuate, inhibit,prevent, or otherwise treat pathogenic infections or neoplastic disease.The pharmaceutical compositions should contain a therapeutic orprophylactic amount of at least one hybrid molecule according to thepresent invention present in a pharmaceutically-acceptable carrier. Thepharmaceutically-acceptable carrier can be any compatible, non-toxicsubstance suitable to deliver the hybrid molecules to an intended host.Sterile water, alcohol, fats, waxes, and inert solids may be used as thecarrier. Pharmaceutically-acceptable adjuvants, buffering agents,dispersing agents, and the like, may also be incorporated into thepharmaceutical compositions. The preparation of pharmaceuticalcompositions incorporating active agents is well described in themedical and scientific literature, see, for example, Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 16th Ed.,1982, the disclosure of which is incorporated herein by reference.

The pharmaceutical compositions just described are suitable for systemicadministration to the host, including both parental and oraladministration. Preferably, the pharmaceutical compositions will beadministered parenterally, i.e., subcutaneously, intramuscularly, orintravenously. Thus, the present invention provides compositions foradministration to a host, where the compositions comprise apharmaceutically-acceptable solution of the hybrid molecules in anacceptable carrier, as described above.

The concentration of the hybrid molecules in the pharmaceuticalcompositions may vary widely, i.e., from less than about 0.1 % by weightof the pharmaceutical composition to about 20% by weight, or greater. Atypical pharmaceutical composition for intramuscular injection would bemade up to contain, for example, 1 to 4 ml of sterile buffered water and1 μg to 1 mg of the hybrid molecule in the present invention. A typicalcomposition for intravenous infusion could be made up to contain 100 to500 ml of sterile Ringer's solution and about 1 to 100 mg of the hybridmolecule.

The pharmaceutical compositions of the present invention can beadministered for prophylactic and/or therapeutic treatment of pathogenicinfection. In therapeutic applications, the pharmaceutical compositionsare administered to a host already infected with the pathogen. Thepharmaceutical compositions will be administered in an amount sufficientto bind to at least a substantial portion of the population of viablepathogens present in the host. An amount adequate to accomplish this isdefined as a "therapeutically effective dose." Such effective dose willdepend on the severity of the infection and on the general state of thepatient's own immune system, but will generally range from about 0.01 μgto 10 mg of the hybrid molecule per kilogram of body weight of the host,with dosages of from about 0.1 μg to 1 mg/kg being more commonlyemployed. In life-threatening situations, it may be desirable toadminister dosages substantially exceeding those set forth above.

For prophylactic applications, the pharmaceutical compositions of thepresent invention are administered to a host not already infected by thepathogen, but perhaps recently exposed to or thought to have beenexposed to, or at risk of being exposed to the pathogen. The hybridmolecules will then be able to block initial infection of the patientcells by the pathogen as well as being able to elicit an immune responsedirectly against the pathogen which may be present. The amount of hybridmolecule required for this purpose, referred to as aprophylactically-effective dosage, are generally the same as describedabove for therapeutic treatment.

For the treatment of neoplastic disease, the pharmaceutical compositionsmay be formulated generally as described above. The dosages andfrequency of administration will depend heavily on the stage of disease,the prognosis, evidence of metastasis, and the like. Frequently,treatment will be performed in combination with other modalities, suchas surgery, radiation treatment, administration of otherchemotherapeutic drugs, and the like.

The following examples are offered by way of illustration, not by way oflimitation.

EXPERIMENTAL

C-glycosides were synthesized as outlined in FIGS. 1-4. Methyl(2,3,4,6-tetra-O-benzyl)-α-D-mannopyranoside 1 was treated withallyltrimethylsilane in acetonitrile using trimethylsilyl triflate(TMSOTf) as a catalyst according to conditions reported by Hosomi et al.(1984) Tetrahedron Lett. 25:2383; Hosome et al. (1987) CarbohydrateResearch 171:223 (FIG. 1). The C-glycosides 2 and 3 were obtained in agreater than 15:1 mixture in an overall yield of 91%. Compound 2 wasdeprotected and reduced by hydrogenolysis (H₂, Pd/C) to give alkane 4(FIG. 2.).

Compounds 6-8 were synthesized from alcohol 5 which was obtained byhydroboration (9-BBN) of compound 2. Oxidation of alcohol 5 using Jones'reagent (CrO₃, H₂ SO₄) followed by hydrogenolysis (H₂, Pd/C) gave thefree acid 6. The amine hydrochloride salt 7 was synthesized fromcompound 5 by mesylation of the primary alcohol (MsCl, Et₃ N) followedby azide displacement (nBu₄ NN₃, CH₃ CN) (Brandstrom et al. (1974) ActaChem. Scand. B 28:699) and hydrogenolysis (H₂, Pd(OH)₂, HCl).Debenzylation (H₂, Pd/C) of compound 5 directly gave alcohol 8. Reactionof compound 7 with N-hydroxysuccinimidobiotin and Et₃ N in 1:1 DMF/MeOHgave conjugate 9 (FIG. 3).

It has been reported that β-O-glycosides of mannose do not bind to E.coli type 1 pili receptors (Firon et al. (1984) Infection and Immunity43:1088). Therefore, as a control for the cell-surface binding studies,we synthesized by β-C-glycoside 3 using a modification of a proceduredeveloped by Lewis et al. (FIG. 4) (Lewis et al. (1982) J. Am. Chem.Sic. 104:4976). The addition of allylmagnesium bromide to lactone 10(Lactone 10 was synthesized from methyl(2,3,4,6-tetra-O-benzyl)-α-D-mannopyranoside by hydrolysis of the methylglycoside (AcOH, H₂ O) followed by Jones' oxidation (CrO₃ /H₂ SO₄) inacetone.) gave hemiketal 11 as a mixture of anomers. Stereoselectivereduction using triethylsilane and boron trifluoride etherate (Et₂ SiH,BF₃ OEt₂) in acetonitrile gave a 1:10 mixture of C-glycosides 2 and 3.Compound 3 was deprotected and reduced by hydrogenolysis to give alkane12.

Compounds 4, 6-9 and 12 were assayed for bacterial receptor bindingusing agglutination studies with yeast cells and their inhibitoryactivity was compared to that of methyl α-D-mannopyranoside 13 (Firon etal. (1983) Carbohydrate Research 120:235; Eshdat et al. (1978) Biochem.Biophys. Res. Commun. 85:1551; and Firon et al. (1982) Biochem. Biophys.Res. Commun. 105:1426. The bacterial strain used in our study was asystemically invasive E. coli K1 pilA+::tetR strain that is responsiblefor sepsis and meningitis in human infants (Bloch et al. (1990)Infection and Immunity 58:275). A summary of the results is given inTable 2.

                                      TABLE 2                                     __________________________________________________________________________    Inhibitory Activity of C-Glycosides of Mannose on the Bacterial               Receptor-Mediated Agglutination of Yeast Cells..sup.a                         Entry                                                                              Compound                         Concentration (mM).sup.b                                                                 Relative Inhibitory                                                           Activity.sup.c               __________________________________________________________________________          ##STR1##                        67          1                           2                                                                                   ##STR2##                        47         1.4                          3                                                                                   ##STR3##                        40         1.7                          4                                                                                   ##STR4##                        13         5.2                          5                                                                                   ##STR5##                        7          9.6                          6                                                                                   ##STR6##                        7          9.6                          7                                                                                   ##STR7##                        1.6        42                           8                                                                                   ##STR8##                        0.6 mM.sup.d                                                                             --                           9                                                                                   ##STR9##                        0.05                                                                              mM (50 μM)                                                                        1340                         __________________________________________________________________________     .sup.a E. coli K1 pilA+::tetR were grown for 24 h at 37° C. on         solid LB media supplemented with tetracycline and were suspended with a       cotton swab in 4 mL of Dulbecco's PBS to a final dilution of 2 ×        10.sup.8 cells/mL. Yeast (Saccharomyces cerevisiae, wild type) were grown     for 36 h on solid YPD media at 30° C. and were suspended with a        cotton swab in 4 mL of Dulbecco's PBS to a final dilution of 1 ×        10.sup.8 cells mL. Protein concentrations were determined by BCA Protein      Assay (Pierce). Agglutination assays were performed on a 20 well ceramic      ring plate. Typically, 90 μl of a solution of the test compound was        combined with 30 μl of the bacterial suspension. After 30 seconds, 30      μl of the yeast suspension was added to give a final volume of 150         μl and the wells were allowed to develop for 3 min. with agitation. A      μl aliquot was removed from each well and spread onto a standard           microscope slide. The slides were quickly heat fixed and mounted with 10      μl of glycerol. The slides were examined under phase contrast at           500× magnification using a Zeiss Axioskip microscope. Agglutination     was observed as clusters of cells. total inhibition of agglutination was      determined by the observation of single cells only.                           .sup.b Concentration causing total inhibition of yeast agglutination.         .sup.c These numbers represent the concentration of methyl                    α-Dmannopyranoside divided by the concentrations listed in column 3     .sup.d Only partial inhibition of agglutination was achieved at this          concentration.                                                           

Three important conclusions can be drawn from these data: (1)Carbon-linked glycosides bind to bacterial mannose lectines and inhibitthe attachment of E. coli K1cells to yeast. Since the 28 kD lectin ishighly conserved in its morphology as determined by its cross reactivitywith monoclonal and polyclonal antibodies (Hanson et al. (1988) Nature332:265), these compounds should also bind to other type 1 pilireceptors. The β-C-glycoside 12 shows no inhibitory activity at aconcentration of 100 mM, demonstrating that the α-specificity of thereceptor observed with O-glycoisides is maintained among C-glycosides.(2) The binding of C-glycosides is stronger than that of methylα-D-mannopyranoside 13. The increase in binding affinity seems to be afunction of the hydrophobicity of the carbon-linked side chain. Forexample, compare the charged compounds 6 and 7 (entries 2 and 3) withthe neutral, hydrophobic compounds 4 and 9 (entries 5 and 6). Compounds4 and 9 inhibit agglutination at a concentration that is approximatelyone order of magnitude less than that of the charged compounds 6 and 7.It is believed that the poor solvation of the hydrophobic side chain inwater increases the affinity of compounds 4 and 9 for the relativelyhydrophobic receptor binding site. This "hydrophobic effect" has alsobeen observed with oxygen-linked glycosides such as compound 14 (entry7) (Firon et al. (1982) supra.).

Finally, it was observed an increase in binding affinity for thebiotin-streptavidin (avidin) system (entries 8 and 9, Table 2).Unfortunately, streptavidin has limited solubility under the conditionsof the assay. Despite the apparent increase in affinity of the conjugateof streptavidin and compound 9, total inhibition was not achieved atthis concentration (entry 8). Avidin, a more soluble protein, wastherefore used in place of streptavidin. It was observed that avidinalone has an intrinsic affinity for the receptor binding site which isprobably due to its glycosylation pattern (total inhibition ofagglutination by avidin is achieved at a concentration of 0.4 mM (Huanget al. (1971) J. Biol. Chem. 246:686)). However, the conjugate of avidinwith compound 9 (entry 9) inhibits agglutination at a concentration of0.05 mM (50 μM), an order of magnitude less than avidin alone, and wasthe tightest binding C-glycoside conjugate in our study. This effect wasbelieved to be due to the tetravalency of the biotin-avidin complex.Biotinylation of ligands that bind to cell-surface receptors can be usedas a general approach to create multivalent ligand arrays with controlover their spacial arrangement.

The biotin-avidin system also allowed targeting molecules to the surfaceof pathogenic organisms. Since the binding of the conjugate alters theantigenic properties of the bacterial surface, the strategy can be usedto target anti-avidin antibodies to the pathogen that would nototherwise recognize the organism.

E coli cells bound with avidin, as described above, were sequentiallyexposed to anti-avidin antibodies and gold (15 nm) colloidal particlesbound to protein A. It was expected that the gold would localize on theE. coli cell surface through an antibody-protein A bridge. Localizationon bacterial pili was confirmed by transmission electron microscopy(TEM). The results are shown in FIG. 5 (typical gold particle shown byarrow) and FIG. 6 (control where E. coli cells incubated with proteinA-gold without anti-avidin antibody).

Binding of complement (Clq) to anti-avidin (IgG2) bound to E. colithrough the biotin-mannose hybrid molecule has also been shown. Clqbinding is the first step in the complement cascase which is part of thehumoral response.

A stable non-hydrolyzable analog of sialic acid 13 (FIG. 7) wassynthesized as follows. The compound is a carbon glycoside of NeuAcprepared by a combined chemical enzymatic approach.

Several methods for the synthesis of carbon glycoside (C-glycosides)have been reported. See, Crich and Lim, (1990) Tet. Lett. 31:1897;Nicotra et al. (1987) J. Org. Chem. 52:5627; Hosomi et al. (1984) Tet.Lett. 25:2383; and Norbeck et al. (1987) J. Org. Chem. 52:2174. In ourhands, many of these methods failed to give C-glycosides of N-acetylneuraminic acid under a variety of conditions. Problems associated withLewis-acid catalyzed methods exist due to the carboxylate. Approachesthat require basic conditions fail because of a variety of unwanted sidereactions. The use of radical coupling reactions eliminates theseproblems.

The key radical species required for C-glycoside synthesis is generatedunder mild conditions and on a carbon atom that can be stabilized by theadjacent carboxylate group and oxygen atom attached to it (capadativeradical). Schmidt et al., for example, has shown thatmethyl-2-deoxy-2-β-chloro-4,7,8,9-tetra-O-acetyl-N-acetyl-neuraminatecan readily be reduced using tributyl tin hydride (Schmidt et al. (1988)Tet. Lett. ₋₋ :3643. Towards this end the ethyl ester of neuraminic acid(Compound 4) was synthesized by an enzyme catalyzed aldol reaction usingNeuAc aldolase between N-acetyl mannosamine (Hosomi et al. (1984) Tet.Lett. 25:2382) and sodium pyruvate to give NeuAc 3 (Bednarski et al.(1988) J. Amer. Chem. Soc. 110:7159; Auge et al. (1984) Tet. Lett.25:4663; and Kim et al. (1988)). Treatment of the crude reaction mixturewith hydrogen chloride gas in ethanol gives the ethyl ester which can bepurified by silica gel chromatography. The data for this compound hasbeen reported in Eschenfelder and Brossmer (1975), Tet. Lett. 35:3069.Treatment of compound (Norbeck et al. (1987) J. Org. Chem. 52:2174) withacetyl chloride at room temperature for 24 h gives the glycosyl chloride16.

Compound 17 was then treated with allyl tributyltin and a catalyticamount of bis (tributyltin) and photolyzed for 18 h using a 450 WattHanovia lamp with a pyrex filter to give approximately a 1:1 mixture ofthe C-glycosides 18 and 19 which were deprotected using sodium ethoxidein aqueous ethanol yielding 19 and 20 which could easily be separated bysilica gel chromatography. Compound 19 (the less polar isomer) isassigned to be the α-anomer in which the carboxylate group is axial;compound 20 (the more polar isomer) is assigned to be the β-anomer. Thestereochemical assignment is based on analogous compounds and NOEstudies.

Detailed synthesis methods for each of the compounds described in theExperimental section are contained in the Appendix to this application.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for introducing a heterologous antigenicdeterminant to the surface of a pathogen or cell, said method comprisingexposing the pathogen or cell to hybrid molecules having a carbohydratebinding moiety which binds specifically to a conserved pathogen orcellular receptor and an effector moiety which includes the heterologousantigenic determinant, wherein the heterologous antigenic determinant isexposed when the binding moiety is bound to the receptor and wherein thehybrid molecule has a molecular weight below about 3 kD.
 2. A method asin claim 1, wherein the carbohydrate binding moiety terminates in atleast one carbon-linked sugar.
 3. A method as in claim 2, wherein thecarbon-linked sugar is selected from the group consisting of mannose,sialic acid, galactose, fucose, α-glucoseamine, galactosamine, andderivatives thereof.
 4. A method as in claim 2, wherein the receptor isa conserved pathogen receptor which mediates attachment of the pathogento susceptible cells.
 5. A method as in claim 1, wherein the receptor isa conserved cellular receptor which mediates metastatic attachment of aneoplastic cell.
 6. A method as in claim 1, wherein the pathogen is aviral pathogen selected from the group consisting of influenza viruses,picornaviruses, and papilloma viruses.
 7. A method as in claim 1,wherein the pathogen is a bacteria selected from the group consisting ofEscherichia coli, and Vibrio cholerae.
 8. A method as in claim 1,wherein the pathogen is a protozoa selected from the group consisting ofEntamoeba histolytica, Plasmodium knowlesi, Plasmodium vivax, andTrypanosoma cruzii.
 9. A method as in claim 1, wherein the bindingmoiety will bind to the pathogen with an affinity of at least about 1mM⁻¹.
 10. A method as in claim 1, wherein the hybrid molecule furthercomprises a linking region which joins the carbohydrate binding moietyto the effector moiety.
 11. A method as in claim 10, wherein the linkingregion is water soluble and has a length in the range from about 10 Å to40 Å.
 12. A method for introducing a heterologous antigenic determinantto a lectin on the surface of a pathogen, said methodcomprising:exposing the pathogen to hybrid molecules including (1) acarbohydrate binding moiety which terminates in at least onecarbon-linked sugar and which binds specifically to the surface lectin,(2) an effector moiety which includes the heterologous antigenicdeterminant, wherein the determinant is exposed when the binding moietyis bound to the lectin, and (3) a linker region which joins thecarbohydrate binding moiety to the effector moiety, wherein the linkerregion is water soluble and substantially free from non-specificcellular binding.
 13. A method as in claim 12, wherein the hybridmolecule has a molecular weight below about 3 kD.
 14. A method as inclaim 12, wherein the carbohydrate binding moiety binds to the surfacelectin with a affinity of at least about 1 mM⁻¹.
 15. A method as inclaim 12, wherein the carbon-linked sugar is selected from the groupconsisting of mannose, sialic acid, galactose, fucose, α-glucoseamine,galactosamine, and derivatives thereof.
 16. A method as in claim 12,wherein effector moiety has a molecular weight below about 1000 D.
 17. Amethod as in claim 16, wherein the effector moiety is selected from thegroup consisting of blood group carbohydrates, dinitrophenol, andGalα1→3Gal.
 18. A method as in claim 17, wherein the binding moiety issialic acid and the effector moiety is Galα1→3Gal.
 19. A method as inclaim 12, wherein the linking region has a length in the range from 10 Åto 40 Å.